Detection Of Duchenne Muscular Dystrophy Carrier By Fluorescence Quantitative Polymerase Chain Reaction

Objective: To develop a method of fluorescence quantitative PCR to detect carrier in the families of Duchenne Muscular Dystrophy associated deletions. Methods:(1)We developed the fluorescence quantitative PCR which is composed of the method of dual PCR ,the fluorescent labeling strategy and the use of dosage quotient analysis. (2)The fluorescence quantitative PCR diagnosed the carriers of DMD deletion families requires several steps: first, the PCR with 9 pairs of primer was used to identify possible deleted exons in the patients with DMD.Second,two pairs of primers for test exon(the deleted )and control exon (the nondeleted) were used to coamplified the DNA of at risk 21 female relatives and normal female controls by using PCR at low cycle number(23 cycles).It was verified that under 23 cycles the assay remains within the exponential phase of amplification.Third, PCR products staining by SYBR GREENⅠ were quantitatively analyzed on densitometric scans of UVP Gel Documentation system.Then,the amount of product amplified from test exon is divided by that from control exon ,and this ratio is calculated with those from controlsamples to obtain a series of dosage quotients(DQ).(3) The normal range of dosage estimates was computed by statistical analysis of normal dosage quotient.We determine the carries status by the normal range of dosage quotient (4) Parts of the results were compared with CK value and pedigree analysis.Results: The ±S of normal female's dosage quotient was 0.5208±0.0524 , The ±S of carrier's dosage quotient was 0.9882±0.0688. 15 female relatives in deleted familes were determined(including 1 obligated deletion carriers ,11presumptive carriers and 3 possible carriers ). 5 presumptive carriers and 1 possible carriers were excluded as the cariers.Conclusions: (1) FQ-PCR is more accurate than the methods based on CK value and pedigree analysis. (2) The fluorescence quantitative PCR is a effective,safe,precise method to detect deleted DMD carriers...